本文選題：netrin-1 + 慢病毒　； 參考：《中南大學(xué)》2011年博士論文

【摘要】：第一章netrin-1與VEGF在糖尿病視網(wǎng)膜病變患者玻璃體腔內(nèi)表達水平變化及關(guān)系 目的檢測增殖期糖尿病視網(wǎng)膜病變(Proliferative diabetic retinopathyPDR)及非糖尿病視網(wǎng)膜病變(Non-diabetic retinopathy NDRP)患者血清及玻璃體腔內(nèi)netrin-1與VEGF的水平,并對PDR患者玻璃體腔內(nèi)netrin-1與玻璃體腔內(nèi)的VEGF表達水平進行相關(guān)性分析,探索研究netrin-1在增殖期糖尿病視網(wǎng)膜病變新生血管形成過程中可能的作用。 方法共18例患者18只眼納入該研究,其中10例為PDR患者(PDR組),8例為NDRP患者(對照組)。于后部玻璃體切割術(shù)中收集未經(jīng)稀釋的玻璃體,并抽取適量血液標(biāo)本,應(yīng)用酶聯(lián)免疫吸附試驗方法檢測玻璃體及血清中netrin-1及VEGF水平。兩組間采用Mann-Whitney U檢驗進行統(tǒng)計學(xué)分析,netrin-1與VEGF水平相關(guān)性采用Spearman's軼和相關(guān)性分析。 結(jié)果PDR患者玻璃體腔內(nèi)netrin-1及VEGF水平均較對照組NDRP患者明顯增高,分別為509.94±138.85vs.85.91±11.72pg/ml,p0.001；762.60±143.14vs.77.52±7.75pg/ml,p0.001。但升高的netrin-1與VEGF水平無明顯相關(guān)性(rho=0.200；P=0.704)。 結(jié)論Netrin-1與VEGF水平在PDR患者玻璃體中均明顯增高,而且,netrin-1可能獨立于VEGF在新生血管形成中起著重要的作用,并可能成為PDR的治療靶點之一。 第二章慢病毒介導(dǎo)的靶向netrin-1的小發(fā)夾狀RNA的病毒構(gòu)建包裝及體外轉(zhuǎn)染篩選 目的構(gòu)建PGCSIL-GFP'慢病毒介導(dǎo)的netrin-1小發(fā)夾狀RNA(small hairpin RNA shRNA)及陰性對照組病毒顆粒。 方法構(gòu)建含netrin-1shRNA序列和陰性對照Scramble shRNA序列的慢病毒載體。將慢病毒載體與pHelper1.0載體、pHelper2.0載體共轉(zhuǎn)染293T細胞,轉(zhuǎn)染后8h更換為完全培養(yǎng)基,培養(yǎng)48h后,收集富含慢病毒顆粒的細胞上清液,并對其濃縮而得到高滴度的慢病毒濃縮液。將所獲得的慢病毒濃縮液體外轉(zhuǎn)染小鼠腦微血管內(nèi)皮細胞(bEnd.3),確定其最佳轉(zhuǎn)染條件。再以同法轉(zhuǎn)染bEnd.3細胞,72h后提取細胞總蛋白,western-blot檢測netrin-1蛋白水平表達變化。 結(jié)果成功構(gòu)建并包裝靶向netrin-1shRNA和陰性對照組shRNA的慢病毒顆粒。與陰性對照組相比,慢病毒介導(dǎo)的netrin-1shRNA體外轉(zhuǎn)染bEnd.3細胞可成功抑制細胞內(nèi)netrin-1的表達。 結(jié)論慢病毒介導(dǎo)的netrin-1shRNA體外轉(zhuǎn)染bEnd.3可成功抑制細胞內(nèi)netrin-1的表達。 第三章慢病毒介導(dǎo)的靶向netrin-1的小發(fā)夾狀RNA抑制視網(wǎng)膜新生血管形成的研究 目的檢測netrin-1在缺氧誘導(dǎo)模型小鼠視網(wǎng)膜中netrin-1的表達,玻璃體腔內(nèi)注射慢病毒介導(dǎo)的netrin-1shRNA抑制netrin-1在視網(wǎng)膜中的表達,并觀察其在病理性視網(wǎng)膜新生血管形成中的作用。 方法48只鼠齡為7天的C57BL/6J小鼠置于濃度為(75±2)%氧艙喂養(yǎng)5天后返回正常氧環(huán)境中。其中36只小鼠于出氧艙后一只眼玻璃體腔內(nèi)注射1μ1慢病毒介導(dǎo)的靶向netrin-1的shRNA (5*105TU)病毒顆粒,對側(cè)眼注射1μ1陰性對照(5*105TU)病毒顆粒。6只不作注射的高氧誘導(dǎo)小鼠作為模型組。另取6只小鼠于正常氧環(huán)境下飼養(yǎng)作為正常對照組。采用real-time PCR、Western blot等方法檢測視網(wǎng)膜中netrin-1mRNA及蛋白質(zhì)的表達水平。FITC-Dextran左心室造影視網(wǎng)膜鋪片了解視網(wǎng)膜血管形態(tài)的改變,組織學(xué)切片并行H-E染色觀察突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細胞核數(shù)量。 結(jié)果：Netrin-1在P17氧誘導(dǎo)視網(wǎng)膜病變小鼠視網(wǎng)膜中的mRNA及蛋白表達水平較正常同齡對照組均明顯增高。玻璃體腔內(nèi)注射特異性慢病毒介導(dǎo)的netrin-1shRNA可有效抑制視網(wǎng)膜中netrin-1的表達,并可減少視網(wǎng)膜新生血管滲漏及視網(wǎng)膜血管無灌注區(qū)的形成,同時,慢病毒介導(dǎo)的netrin-1shRNA用藥組突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細胞核數(shù)量為9.8±1.7,較同齡scramble shRNA轉(zhuǎn)染組對照組(20.3±3.4)明顯減少約50%以上(P0.05)。 結(jié)論netrin-1在缺氧誘導(dǎo)的小鼠視網(wǎng)膜病變中表達上調(diào),慢病毒介導(dǎo)的靶向netrin-1的shRNA可有效抑制視網(wǎng)膜新生血管形成,為血管增殖性視網(wǎng)膜病變的治療提供了新的靶點。
[Abstract]:Chapter one netrin-1 and VEGF expression levels in vitreous cavity of patients with diabetic retinopathy and their relationship
Objective to detect the levels of netrin-1 and VEGF in serum and vitreous cavities of patients with proliferative diabetic retinopathy (Proliferative diabetic retinopathyPDR) and non diabetic retinopathy (Non-diabetic retinopathy NDRP), and to analyze the correlation of VEGF expression in the cavity of netrin-1 and glass in the vitreous cavity of patients with PDR. Objective to explore the possible role of netrin-1 in neovascularization of proliferative diabetic retinopathy.
Methods a total of 18 patients with 18 eyes were included in this study, of which 10 were PDR patients (group PDR) and 8 were NDRP patients (control group). In the posterior vitrectomy, the undiluted vitreous body was collected and a proper amount of blood samples were extracted. The level of netrin-1 and VEGF in glass body and serum was detected by enzyme linked immunosorbent assay. The two groups were used to Mann-. Whitney U test was used for statistical analysis. The correlation between netrin-1 and VEGF level was analyzed by Spearman's and correlation analysis.
Results the levels of netrin-1 and VEGF in the vitreous cavity of PDR patients were significantly higher than those of the control group NDRP, respectively, 509.94 + 138.85vs.85.91 + 11.72pg/ml, p0.001, and 762.60 + 143.14vs.77.52 + 7.75pg/ml, but there was no significant correlation between netrin-1 and VEGF level.
Conclusion the level of Netrin-1 and VEGF is significantly higher in the vitreous of PDR patients. Moreover, netrin-1 may be independent of VEGF in the formation of neovascularization, and may be one of the targets for the treatment of PDR.
The second chapter is the construction and packaging of lentivirus mediated small hairpin RNA targeting netrin-1 and screening in vitro.
Objective to construct PGCSIL-GFP'lentivirus mediated netrin-1 hairpin RNA (small hairpin RNA shRNA) and negative control group virus particles.
Methods the lentivirus vector containing the netrin-1shRNA sequence and the negative control Scramble shRNA sequence was constructed. The lentivirus vector was co transfected with the pHelper1.0 vector and pHelper2.0 vector, and the 293T cells were transfected with the pHelper2.0 vector. After the transfection, the 8h was replaced as a complete medium. After the culture of 48h, the cell supernatant rich in lentivirus particles was collected and the high titer was obtained. The concentration liquid was transfected into the mouse brain microvascular endothelial cells (bEnd.3), and the optimal transfection condition was determined. The bEnd.3 cells were transfected with the same method, the total protein of the cell was extracted after 72h, and the expression of netrin-1 protein was detected by Western-blot.
Results the lentivirus particles targeting netrin-1shRNA and negative control group shRNA were successfully constructed and packaged. Compared with the negative control group, the transfection of bEnd.3 cells with netrin-1shRNA in vitro could inhibit the expression of netrin-1 in the cell successfully.
Conclusion lentivirus mediated netrin-1shRNA transfection in vitro can inhibit the expression of netrin-1 in bEnd.3 cells.
The third chapter lentivirus mediated small hairpin RNA targeting netrin-1 inhibits retinal neovascularization.
Objective to detect the expression of netrin-1 in the retina of hypoxic induced model mice, and to observe the role of netrin-1 in retinal neovascularization by intravitreal injection of lentivirus mediated netrin-1shRNA to inhibit the expression of netrin-1 in the retina.
Methods 48 C57BL/6J mice aged 7 days were placed in the normal oxygen environment after feeding for 5 days with a concentration of (75 + 2)% oxygen chamber. 36 mice were injected with 1 mu 1 lentivirus mediated shRNA (5*105TU) virus particles in the posterior vitreous cavity of the oxygen chamber and injected with 1 micron negative control (5*105TU) virus particles.6 only on the lateral eye. Mice were injected with hyperoxia as model group. Another 6 mice were taken as normal control group under normal oxygen environment. The expression of netrin-1mRNA and protein in retina was detected by real-time PCR, Western blot and so on..FITC-Dextran left ventriculography retina spread was used to understand the changes of retinal vascular morphology and histology. Slice parallel H-E staining was used to observe the number of endothelial cells that broke through the inner limiting membrane of the retina.
Results: the expression level of mRNA and protein in the retina of P17 oxygen induced retinopathy of mice was significantly higher than that of the normal control group. Intravitreal injection of specific lentivirus mediated netrin-1shRNA could effectively inhibit the expression of netrin-1 in the retina, and reduce the retinal neovascularization and retinal vascular failure in the retina. At the same time, the number of vascular endothelial nuclei of the lentivirus mediated netrin-1shRNA group broke through the inner boundary membrane of the retina was 9.8 + 1.7, compared with that of the scramble shRNA transfection group (20.3 + 3.4) of the same age group (20.3 + 3.4), which was significantly reduced by more than 50% (P0.05).
Conclusion the expression of netrin-1 is up-regulated in the retinopathy of hypoxia induced mouse retinopathy. The shRNA targeting netrin-1 mediated by lentivirus can effectively inhibit the formation of retinal neovascularization, and provides a new target for the treatment of vascular proliferative retinopathy.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R739.72

【共引文獻】

相關(guān)期刊論文 前4條

1 宋愛琴;鞠秀麗;孫立榮;王玲珍;李曉玲;于洪升;;慢病毒載體介導(dǎo)的siRNA抑制Jiyoye細胞c-myc基因表達的實驗研究[J];山東大學(xué)學(xué)報(醫(yī)學(xué)版);2011年04期

2 熊思齊;夏曉波;蔣劍;孫偉;;軸突導(dǎo)向因子-1 mRNA在氧誘導(dǎo)血管增生性視網(wǎng)膜病變中的表達[J];眼科研究;2009年02期

3 劉丹;黃q，

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